Figure 1.
An Inhibitor of CD38, CZ-48 Induces Intracellular cADPR Production
(A) Wild-type and CD38-EGFP-overexpressing HEK-293T cells were treated with 100 μM CZ-48 for 24 h, and cADPR contents were analyzed by cycling assay.
(B) The target compound was separated by HPLC. HEK-293T cells were treated with 100 μM CZ-48 for 72 h, and the nucleotides were extracted and fractionated by HPLC with an AG MP-1 column (blue line, left y axis). Fractions 4, 5, and 6 (Peak 13, green box) showed positive signals in the cycling assay (red line, right y axis).
(C) Peak 13 released Ca2+ from sea urchin homogenate similar to 0.5 μM cADPR, was blocked by 500 μM 8Br-cADPR pre-treatment of the homogenate, and was destroyed by 10 μg/mL reCD38 pre-treatment of the compound.
(D) Peak 13 produced the fluorescence signals similar to 0.5 μM cADPR in cycling assay.
(E) The time course of cADPR production in the CZ-48-treated HEK-293T cells. HEK-293T cells were treated with 100 μM CZ-48 for different time periods, and cADPR contents were analyzed by cycling assay.
(F) Dose-response curves of CZ-48 in intracellular cADPR production and NAD consumption. HEK-293T cells were treated with different doses of CZ-48 for 24 h, and the amounts of cADPR and NAD were analyzed. All the above-mentioned experiments were repeated at least three times (means ± SDs; n = 3).